The long-term objective of this research program is to determine how progestins modulate gene expression, with emphasis on the mechanism of action of progesterone receptors (PRs) in regulating gene transcription. Two isoforms of PR have previously been identified: A- and B-receptors. The investigator and her colleagues have recently identified a third progesterone receptor, termed the C-receptor. Based on mRNA mapping studies, the C-receptor lacks the N-terminus and the first DNA binding finger of A- and B-receptors, but contains the second DNA-binding finger, the hormone binding domain, the nuclear localization signal, and sequences for dimerization. Since the C-receptor lacks the first DNA- binding finger, the investigator predicts that it does not bind DNA and, by itself, would be transcriptionally inactive. The working hypothesis of this application is that C-receptors negatively modulate progestin action by dimerization with active A- and B-receptors. Aim #1 is to determine if utilization of an alternate downstream start site (Met 595) in the human PR cDNA results in the synthesis of C-receptors. This will be accomplished by generating synthetic C-receptors in an in vitro transcription-translation system from a truncated human PR cDNA lacking all upstream start sites but containing Met 595. Aim #2 is to determine the interactions of C-receptors with A- or B-proteins and assess its effect On the transcriptional activity of A- and B-receptors. This will be done by analyzing the different isoforms in a dimer using antibody co- precipitation techniques and gel retardation assays. The trans- activational capacity of A- and B- receptors will be assessed in transfection assays. Aim #3 is to determine the presence and hormonal regulation of C-receptors in progestin-responsive cells and tissues. This will be accomplished by analyzing cells or tissues for the presence of C-specific mRNA transcriptions and C-receptor proteins, and determining whether C-receptor synthesis is regulated by hormones.